Case series on Trichomonas vaginalis infections: impact of proper sample collection and diagnostic stewardship

This paper elucidates the transformative impact of a strategic shift in diagnostic practices in the detection of Trichomonas vaginalis. It explores five cases where the implementation of a specific diagnostic protocol led to effective identification of the infection. In-depth discussions and a comprehensive literature review underline the necessity for precise diagnosis and the paramount importance of diagnostic stewardship in managing sexually transmitted infections.


InTRODuCTIOn
Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is one of the most common non-viral sexually transmitted infections (STIs) worldwide, affecting millions annually [1].However, its prevalence may be underestimated due to diagnostic inaccuracies often associated with improper sample collection and laboratory methods.
Trichomoniasis remains a significant public health concern.Globally, the World Health Organization (WHO) reported approximately 156 million new cases in 2016.In India, studies suggest that T. vaginalis infection rates range between 8.5-34 % among women attending STI clinics, with higher prevalence in certain regions [2].For instance, in Gujarat, data indicate a prevalence rate of about 34 % (out of 102 samples, 35 were positive for T. vaginalis by the culture method) in women seeking reproductive health services [3].These numbers underline the importance of efficient diagnostic and therapeutic interventions.This paper presents a thorough review of the literature and five case studies, comprising four women and one man, diagnosed with trichomoniasis, highlighting the critical role of improved diagnostic practices in disease detection and management.

LITERATuRE REvIEw On T. vaginalis DIAgnOSTIC APPROAChES
The detection and management of T. vaginalis infections, a leading cause of sexually transmitted infections globally, has seen noteworthy evolution over the decades.As the medical and scientific communities' understanding of the organism's behaviour has grown, so too have the methodologies to detect it.

CuRREnT bEST PRACTICES
The optimal diagnostic approach for T. vaginalis often depends on multiple factors: the clinical setting, available resources, the urgency of diagnosis and the patient's symptoms.Many guidelines now advocate for a combination of methods, using rapid tests or microscopy for immediate patient management while employing NAATs for screening programmes, especially in populations with a high prevalence of asymptomatic carriers.
The journey of T. vaginalis diagnosis reflects the broader evolution of medical science, where traditional methods give way to or are complemented by advanced techniques, all with the shared goal of enhancing patient care.While each method has its merits, the continued exploration of even more accurate, cost-effective and rapid diagnostic tools will always be a priority in the field.

CASE PRESEnTATIOn Case 1
A 28-year-old female presented with complaints of vaginal itching and a malodorous, frothy discharge for the past 5 days.The patient reported no significant medical history and denied any previous STIs.Vaginal examination revealed erythema and inflammation of the vaginal walls.Wet mount microscopy confirmed the presence of motile T. vaginalis trophozoites, thus confirming the diagnosis of trichomoniasis.The patient was prescribed oral metronidazole 500 mg twice daily, and her symptoms resolved after 7 days of treatment.

Case 2
A 35-year-old male presented with urethral discharge and dysuria.The patient reported multiple sexual partners and inconsistent condom use.Urethral swab microscopy revealed motile T. vaginalis trophozoites, indicating trichomoniasis.The patient received a 2 g single dose of oral metronidazole, leading to the resolution of symptoms within a week.

Case 3
A 25-year-old pregnant female in her second trimester was screened for STIs as part of routine antenatal care.Vaginal swab microscopy confirmed the presence of T. vaginalis trophozoites.The patient was treated with 1 % topical metronidazole gel once daily.Follow-up examinations revealed complete resolution of the infection.

Case 4
A 22-year-old woman reported lower abdominal pain, pain during intercourse (dyspareunia), white vaginal discharge and bleeding after intercourse.Microscopy revealed motile T. vaginalis trophozoites, with a vaginal pH of 7. The patient was prescribed oral metronidazole 500 mg twice daily for 7 days.

Case 5
A 46-year-old postmenopausal woman with a history of hysterectomy and asthma presented with itching and burning around the perineum, right flank pain.Her vaginal discharge pH was 6, with sluggishly motile T. vaginalis trophozoites identified.The patient received oral metronidazole 500 mg twice daily for a week.
Cases 4 and 5 were lost to follow-up so the clinical outcome for these patients is unknown.

Clinical presentation and diagnostic approaches
The patients in the case series presented with diverse clinical manifestations that spanned from heavy bleeding and white vaginal discharge to lower abdominal pain, dyspareunia and post-coital bleeding.Such diversity of symptomatology is frequently noted in trichomoniasis cases, as supported by the existing literature [4,6].It is this wide range of manifestations that makes trichomoniasis a clinical chameleon, often leading to misdiagnosis or underdiagnosis.Therefore, ensuring the accuracy of laboratory diagnosis is of paramount importance for the successful management of each patient's condition.

Protocols to trigger testing for T. vaginalis
Given the protean clinical manifestations of T. vaginalis, establishing a definitive protocol for testing is imperative.Our guidelines for triggering a diagnostic test for T. vaginalis encompass a combination of clinical presentations, risk assessments and patientspecific factors.
• Clinical presentation.While asymptomatic cases remain a challenge, commonly encountered symptoms such as vaginal or urethral discharge, itching, pain during intercourse, or post-coital bleeding immediately warrant testing.• Risk assessment.Patients with a history of multiple sexual partners, inconsistent condom use, or known contact with an infected individual are considered high-risk and are thus recommended for testing.• Patient-specific factors.Pregnant women are routinely tested due to the potential complications T. vaginalis can pose during pregnancy.Additionally, individuals with a history of recurrent STIs or those presenting with other STI symptoms are also prioritized for testing to rule out co-infections or a misdiagnosed recurrent infection.• Other triggers.Given its 'clinical chameleon' nature, any unexplained or persistent reproductive tract symptoms, especially if other tests come back negative, serve as a trigger for T. vaginalis testing as a diagnostic precaution.
By adhering to these protocols, we aim to capture a broad spectrum of potential T. vaginalis infections, ensuring that even subtle presentations do not go undetected.
Historically, the laboratory diagnosis of T. vaginalis has been challenging, primarily due to our inability to cultivate the organism and the transient presence of the protozoa in clinical samples.However, microscopic examination of a wet mount is an effective and rapid tool for diagnosing trichomoniasis.It provides direct visualization of the motile trophozoites of T. vaginalis, enabling a swift diagnosis [7].Additionally, the Gram staining technique, a mainstay in microbiology, provides robust diagnostic support, especially in resource-limited settings where advanced diagnostic tools are not readily available.
Importantly, an accurate diagnosis hinges on proper sample collection.Poor collection methods can yield false negatives, leading to missed or delayed diagnosis and treatment.In this series, correct sample collection has been shown to be pivotal in obtaining accurate diagnoses, revealing an opportunity to improve diagnostic protocols.

Shift in diagnostic protocols[8]
Traditionally, our primary diagnostic approach for T. vaginalis revolved around culture-based methods, which, while sensitive, often required prolonged turnaround times and specific conditions for organism viability.Recognizing the need for more immediate results, particularly in acute care scenarios, we transitioned to emphasizing wet mount microscopy.This method, involving the direct microscopic visualization of samples, offers rapid results and a practical solution for on-the-spot clinical decisions.While still retaining culture methods for specific scenarios where they might offer added value, the shift towards wet mount microscopy represents our strategic adaptation to the evolving demands of clinical care, balancing speed with diagnostic accuracy.

Refining sample collection protocols for optimal diagnosis
Accurate diagnosis of T. vaginalis often hinges on the quality of the sample collected.Historically, our sample collection was more generalized and lacked specificity, sometimes leading to compromised sample integrity, especially if not processed immediately.This led to occasional false negatives or inconclusive results.Recognizing this limitation, we introduced rigorous guidelines and training for healthcare personnel involved in sample collection.
The new standard emphasizes the following.
• Timely processing.Ensuring that samples are transported to the laboratory and processed as swiftly as possible to maintain organism viability.
To address timely transportation and processing of samples, our facility implemented a decentralized approach.The microbiology laboratory is situated in close proximity to the outpatient services of the hospital and obstetrics and gynaecology outpatient services are among them.This reduced transit time ensured immediate processing, maintaining organism viability.This setup also alleviated the burden on the main laboratory, allowing for more focused and efficient handling of genitourinary (GU) samples.For ensuring adequate sample volume with swabs, our team focused on training healthcare professionals in proper swabbing techniques.This included the correct insertion depth, duration of contact with the mucosal surface and rotation of the swab to ensure sufficient sample collection.By adhering to these refined sampling standards, we aim to enhance the accuracy of the diagnostic process, ensuring that every sample has the highest potential for yielding a true and representative result.

Co-testing protocols and outcomes
Recognizing the potential for co-infections and the overlapping symptomatology of various STIs with T. vaginalis, we employed a multi-test diagnostic approach in all presented cases, as follows.
• Microscopy for clue cells.To rule out bacterial vaginosis, microscopic examinations were conducted to detect the presence of clue cells.None of the female patients exhibited clue cells on their wet mount microscopy.• Syphilis testing.Given the risk and implications of untreated syphilis, all patients underwent a rapid plasma reagin (RPR) test.All tests returned non-reactive, indicating no active syphilis infections among the patients.• HIV testing.Considering the enhanced risk of HIV transmission associated with STIs, HIV tests were also conducted for each patient.All tested negative.
While our facility does not offer chlamydia and gonorrhoea NAATs, we emphasize clinical evaluation and other available tests to achieve a comprehensive diagnostic picture.This thorough approach ensures an understanding of the broader STI status of the patient, paving the way for informed treatment decisions.

Treatment protocols for T. vaginalis
The choice of treatment regimen for T. vaginalis at our facility is dictated by several factors, including the patient's clinical presentation, co-existing medical conditions and specific circumstances.Below is an overview of our treatment protocols and the rationale for each.
• Standard treatment.The primary treatment for uncomplicated T. vaginalis infection in non-pregnant individuals is a single dose of oral metronidazole.This approach is backed by its high efficacy, convenience for the patient and compliance assurance.• Extended treatment.In cases where symptoms are more severe or persistent, or where there is a history of recent treatment failures, a 7 day regimen of metronidazole is preferred.This extended duration ensures complete eradication of the infection and minimizes the chances of resistance or recurrence.
• Pregnant patients.Guidelines from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend oral metronidazole as the standard treatment for T.s vaginalis in pregnant women [9,10].This recommendation is based on its well-established safety profile and the substantial risks associated with T. vaginalis infections during pregnancy, which include pre-term delivery and low birth weight.Consequently, topical treatment is no longer advocated as the primary therapy due to its lower efficacy than systemic treatment.• Treatment guidelines.Our protocols align with the treatment recommendations set forth by the WHO and the CDC.Both of these guidelines emphasize tailoring the treatment approach based on the individual's clinical and demographic factors [9,10].
By adhering to these standardized protocols and guidelines, we ensure that each patient receives optimal care tailored to their specific needs, ensuring the highest chances of treatment success.

The role of sensitization and training
Our efforts to improve diagnosis started with a sensitization and training session among healthcare providers.This focused on proper sample collection and reducing unnecessary requests for cultures.As a result, our laboratory observed an improvement in diagnostic practices, leading to better patient management.This outcome aligns with numerous studies that have highlighted the positive impact of similar training interventions on improving clinical-laboratory coordination and thereby enhancing patient outcomes [11].
Such sensitization and training efforts are critical to improving the diagnostic capabilities of health providers.In our cases, not only did these efforts lead to the diagnosis of five new cases of trichomoniasis, but they also contributed to a larger shift in diagnostic strategies, emphasizing the judicious use of diagnostic tools and a deeper understanding of their clinical applications.

Training intervention and its transformative impact
To address recurring challenges in sample quality and unnecessary test requisitions, we orchestrated a targeted interactive session with our obstetrics and gynaecology colleagues in May 2023.This initiative was driven by the increasing influx of high vaginal swabs (HVSs) without discernible clinical relevance.

Content of the training
Our training was structured around key themes to ensure a comprehensive understanding.
• Proper sample collection.Emphasized the critical role of obtaining high-quality samples, discussing the ideal techniques and common pitfalls to avoid.• Wet mount microscopy.Introduced its significance in the rapid and accurate diagnosis of infections like trichomoniasis, illustrating its practical application.• Gram staining.Demonstrated its pivotal role in discerning various infections, particularly in distinguishing between bacterial vaginosis, candidiasis and trichomoniasis.• Questioning the necessity of culture.Given that many causative agents of bacterial vaginosis are anaerobes and both candidiasis and trichomoniasis can be effectively diagnosed with Gram staining and wet mount, we questioned the routine need for culture.This segment emphasized the judicious use of culture, conserving resources and ensuring timely diagnoses.

Post-training impact
The results of this training were multifaceted and immensely positive, with most of our cases reporting positive post-training effects.
• Decrease in unnecessary requests.There was a marked reduction in superfluous culture and sensitivity requests.This not only conserved resources but also ensured that laboratory efforts were channelled into clinically relevant tests.• Enhanced understanding among gynaecologists.The session armed the obstetrics and gynaecology specialists with insights into proper sample collection and relevant test requisition.Their newfound knowledge ensured that samples sent were of optimal quality and that the requested tests aligned with the patient's clinical presentation.• Collaborative synergy.This initiative fostered a collaborative environment, bridging the gap between laboratory specialists and clinicians.This synergy promises better patient outcomes, with both teams being well informed and aligned in their approach.
In essence, through this targeted training session, we successfully optimized our diagnostic approach, ensuring efficient utilization of resources and fostering a collaborative spirit between laboratory specialists and clinicians.

Diagnostic stewardship and its implications
Diagnostic stewardship represents an emerging field of interest, particularly in the face of increasing antimicrobial resistance.This concept emphasizes the need for efficient use of laboratory resources to improve patient outcomes [12].Through the case series presented, we showcase how proper sample collection and the appropriate use of diagnostic tests, such as wet mount and Gram staining, can provide a reliable and cost-effective solution.Moreover, these practices help reduce unnecessary cultures, which aligns with the global antimicrobial stewardship (AMS) goals, thus contributing to the prevention of antimicrobial resistance [5,13].
Diagnostic stewardship is increasingly gaining recognition for its potential to improve patient outcomes and reduce healthcare costs.These benefits are not only limited to patients but also extend to the healthcare system as a whole, by optimizing the use of diagnostic tests, preventing unnecessary treatments and reducing the risk of antimicrobial resistance.
Looking ahead, continuous efforts are needed to further promote the principles of diagnostic stewardship and ensure their implementation at all levels of healthcare.By doing so, we can achieve more accurate and timely diagnoses, which will ultimately contribute to improved patient outcomes and greater effectiveness in our battle against antimicrobial resistance.
Through our case series and the subsequent changes in laboratory diagnostic practices, we emphasize the profound impact of correct sample collection, judicious use of laboratory diagnostic tests and effective sensitization and training efforts.Each of these components plays a crucial role in the accurate diagnosis and successful management of trichomoniasis.

microscopy within diagnostic stewardship
The ethos of diagnostic stewardship is to ensure that the right test is given to the right patient at the right time.Microscopy, especially wet mount microscopy for the detection of T. vaginalis, aligns with these principles in the following ways.
• Rapid and reliable.Microscopy provides an immediate visualization of T. vaginalis trophozoites, offering swift diagnosis.Its accuracy, when performed correctly, ensures that patients receive timely and appropriate treatment.• Cost-effective.Given the resource constraints in many healthcare settings, microscopy serves as an affordable diagnostic tool.
It does not require advanced equipment or reagents, making it accessible and sustainable.• Targeted testing.While our paper may give the impression that microscopy is conducted universally, in practice, it is administered based on clinical judgement.Patients presenting with symptoms consistent with trichomoniasis or those identified as high-risk through our screening protocols are prioritized for microscopic evaluation.• Complement to other tests.In cases where microscopy results are inconclusive or co-infections are suspected, microscopy can act as a stepping stone to more specific tests.It aids in narrowing down differential diagnoses, ensuring that subsequent tests are more focused and are used judiciously.• Reduction in unnecessary treatment.Accurate diagnosis through microscopy reduces the chances of treating non-existent infections.This not only prevents unnecessary drug exposure for patients but also aids in antimicrobial stewardship, reducing the chances of resistance development.
By integrating microscopy within our diagnostic approach, and using it judiciously based on clinical indicators, we align with the principles of diagnostic stewardship.It ensures that our patients receive optimal care, that diagnostics are cost-effective and that resources are utilized efficiently.

global implications and the way forward
Our case series underlines the need for strengthening diagnostic capacities, particularly in resource-limited settings where the disease burden of STIs, including trichomoniasis, is often high.It calls for the development and implementation of more such strategic interventions that can empower healthcare providers with the right knowledge and skills to accurately diagnose and manage these infections [14].
Moreover, our findings have broader implications for the global health agenda, specifically for antimicrobial stewardship (AMS).Unnecessary cultures and consequent inappropriate antibiotic use can contribute to the growing menace of antimicrobial resistance.Thus, efficient diagnostic protocols can directly contribute to AMS objectives.

COnCLuSIOn
In conclusion, this case series and literature review underscores the critical role of diagnostic stewardship in the management of T. vaginalis infections and other sexually transmitted infections.A paradigm shift towards appropriate diagnostic practices, facilitated by sensitization and training of healthcare providers, can result in more accurate diagnoses, better patient management and significant progress towards global AMS goals.
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Find out more and submit your article at microbiologyresearch.org I would expect that as part of timely processing of samples, sometimes a mini side lab equipped with basic microscopy equipment and personnel is deployed to achieve this and also reduce GU samples competing with general lab samples.So how did you achieve timely transportation andprocessing?
196,197•Timely Processing: Ensuring that samples are transported to the laboratory and processed as swiftly as possible to maintain organism viability.

Response:
To address timely transportation and processing of samples, our facility implemented a decentralized approach.The microbiology laboratory is situated in a close proximity to the outpatient services of the hospital and Obstetrics and Gynaecology outpatient services is one amongst them.This reduced transit time and ensured immediate processing, maintaining organism viability.This setup also alleviated the burden on the main laboratory, allowing for more focused and efficient handling of genitourinary (GU) samples.
This paragraph has been added into the revised manuscript from line number 202 to 208.

Comment
How can sample volume be termed adequate in samples taken with swabs?The figure mentioned earlier in the introduction has been rectified and the new figures are added to the manuscript which reflects in the line numbers 43-45.
The above-mentioned reference is added (reference number 2).The reference Das et all 2011 has been removed from the reference section.

Comment
Reference This has been added to the introduction (line number 45 to 47).The previous reference (patel et al 2006) has been removed and the aforementioned reference has been added to the reference section (Reference 3).

Comment
Heading a) Traditional Diagnostic Approaches: Reference 1 (Schwebke et al 2004) does not support suggestion sensitivity of microscopy is as low as 50% -they state 60-70% sensitivity.Please review.

Response:
As appropriately pointed out by the reviewer necessary changes has been made and incorporated into the manuscript which is reflected in the line number 64.

Comment
Heading: case presentation Please specify metronidazole doses used and strength of topical treatments.

Response:
In case presentations where metronidazole is used for the treatment of Trichomonas vaginalis, the doses used were: • For oral treatment: Metronidazole 500 mg twice daily for 7 days for females and Metronidazole 2g single dose for male.
• For topical treatment: Metronidazole gel 5 gram preparation of 1% Metronidazole gel, applied once daily.
This has been added to the manuscript and is reflected in the line numbers 115, 121, 125, 132-133, 137-138.

Comment
In case 3 -please remove the following sentence as it incorrectly implies oral metronidazole is unsafe in pregnancy: "Considering her pregnancy, the patient was treated with topical metronidazole gel to avoid potential risks associated with oral medication".

Response:
As appropriately pointed out by the reviewer, the sentence has been removed from the revised manuscript.

Comment
Cases 4 and 5 have no treatment mentioned.please review.

Response:
The treatment prescribed to cases 4 and 5 has been mentioned in the revised manuscript.Both the patients were lost to follow up.So, the outcome was not known.
This has been mentioned in the revised manuscript in the line numbers 132-133, 137-139.

Comment
Heading: shift in Diagnostic protocols Reference 7 (Ali and Nozaki 2007) is a review article about therapeutics for parasites.It contains nothing about diagnostic processes.Please review.

Response:
The This citation has replaced the previous Reference 7 in the manuscript to accurately reflect current knowledge on diagnostic approaches for Trichomonas vaginalisand is reflected as Reference 7 in the revised manuscript.

Comment:
Heading: Treatment protocols for Trichomonas vaginalis Please state doses used for metronidazole.

Response:
The recommended doses for metronidazole in the treatment of Trichomonas vaginalis, as per CDC guidelines, are: For women: Metronidazole 500 mg orally twice a day for 7 days.
For men: Metronidazole 2 g orally in a single dose .
This has been added to the manuscript and is reflected in the line numbers 115, 121, 125, 132-133, 137-138.

9.Comment
Pregnant Patients: There is no evidence of teratogenicity from use of oral metronidazole during any stage of pregnancy.However, there is evidence that TV causes serious complications in pregnancy such as pre-term delivery and low birth weight.The efficacy of topical therapeutics for TV are uncertain at best -conflicting results in the literature, some suggesting topical metronidazole is inferior to systemic therapy.This is reflected in the WHO guideline you mention whereby they advise topical metronidazole is not recommended for TV and that oral metronidazole is recommended for treatment of TV in pregnancy (Sherrard, IUSTI/WHO management of vaginal discharge 2018).
As such, I cannot condone suggesting to the reader that topical treatment of TV in pregnancy is sufficient.

Response:
Considering the most recent guidelines provided by the Centres for Disease Control and Prevention (CDC) and the World Health Organization (WHO), our manuscript has been updated to accurately reflect that oral metronidazole is the recommended therapeutic approach for treating Trichomonas vaginalisin pregnancy.This recommendation is based on its well-established safety profile and the substantial risks associated with TV infections during pregnancy, which include pre-term delivery and low birth weight.Consequently, topical treatment is no longer advocated as the primary therapy due to its comparatively lower efficacy than systemic treatment.
This has been added to the manuscript and is reflected from line numbers 249-256.

Comment
Please provide references for WHO and CDC guidelines.

Response:
Centres for Disease Control and Prevention.

Response:
The reference number 8 has been removed from the text as well as reference section of the manuscript.We are sorry for the mistake.

Comment
Reference

Please rate the quality of the presentation and structure of the manuscript Very good
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes 1. Whilst it is implied within the manuscript that the key change has been to shift from standard culture to wet mount microscopy, this is not explicitly or clearly described.Please include a paragraph which clearly articulates what the previous standard diagnostic protocols were, and how they have now been changed.
Traditionally, our primary diagnostic approach for T. vaginalis revolved around culture-based methods, which, while sensitive, often required prolonged turnaround times and specific conditions for organism viability.Recognizing the need for more immediate results, particularly in acute care scenarios, we transitioned to emphasizing wet mount microscopy.This method, involving the direct microscopic visualization of samples, offers rapid results and a practical solution for on-the-spot clinical decisions.While still retaining culture methods for specific scenarios where they might offer added value, the shift towards wet mount microscopy represents our strategic adaptation to the evolving demands of clinical care, balancing speed with diagnostic accuracy.
This paragraph has been added into the revised manuscript from line number 179 to 188.
2. Similarly, the manuscript makes repeated reference to improving 'proper sampling', however there is no description as to what the authors mean in relation to sampling standards.How have these changed and what is the new standard?

Refining Sample Collection Protocols for Optimal Diagnosis:
Accurate diagnosis of T. vaginalis often hinges on the quality of the sample collected.Historically, our sample collection was more generalized and lacked specificity, sometimes leading to compromised sample integrity, especially if not processed immediately.This led to occasional false negatives or inconclusive results.Recognizing this limitation, we introduced rigorous guidelines and training for healthcare personnel involved in sample collection.
The new standard emphasizes: •Timely Processing: Ensuring that samples are transported to the laboratory and processed as swiftly as possible to maintain organism viability.
•Correct Sampling Site: Depending on the patient's symptoms and gender, emphasizing the correct anatomical site for sample collection.
•Avoiding Contamination: Ensuring that samples are free from contaminants, including lubricants or douches, which can interfere with microscopy.
•Volume and Technique: Adequate sample volume and correct swabbing technique to ensure a representative sample.
By adhering to these refined sampling standards, we aim to enhance the accuracy of the diagnostic process, ensuring that every sample has the highest potential for yielding a true and representative result.
This has been added to the revised manuscript from line numbers 189 -206.
3. The abstract states the authors have conducted a comprehensive literature review, however this is not apparent in the main body of the manuscript.A comprehensive literature review of TV infections should be performed or at least referenced int he linked literature.
As suggested a literature of review on Trichomonas vaginalis diagnostic approaches has been done and added into the revised manuscript from line numbers 51 to 104.
4. The authors state disease burden is high however offer no epidemiological data to corroborate estimates of TV incidence or prevalence.
Trichomoniasis remains a significant public health concern.Globally, the World Health Organization reported approximately 156 million new cases in 2016.In India, recent studies suggest that T. vaginalis infection rates range between 2% to 10% among women attending STI clinics, with higher prevalence in certain regions.For instance, in Gujarat, data indicates a prevalence rate of about 7% in women seeking reproductive health services.These numbers underline the importance of efficient diagnostic and therapeutic interventions.
This paragraph has been added in the Introduction section of the revised manuscript from line numbers 42 to 47.
5. The introduction states 5 cases of female TV infections will be presented, then 4 females and 1 male are discussed in the main body of the manuscript.
The rectification has been made and added to the revised manuscript from line numbers 48 to 50.
6.It is unclear as to the timescale over which these patients were identified.Please clearly include dates of the cases in order to strengthen your assertion that the diagnoses were a result of changes in diagnostic practices.
The suggested changes has been included in line numbers 262 of our revised manuscript.
7. The authors report that TV is a 'clinical chameleon', insinuating that this diagnosis cannot be reached on the basis of clinical history and examination findings alone.There is a spectrum of clinical presentations reported ranging from asymptomatic carriage to vaginal/urethral discharge and bleeding -please comment on what protocols are in place to trigger testing for TV -is there a specific set of symptoms, risk profile, patient characteristics?

Protocols to Trigger Testing for Trichomonas vaginalis:
Given the protean clinical manifestations of Trichomonas vaginalis, establishing a definitive protocol for testing becomes imperative.Our guidelines for triggering a diagnostic test for T. vaginalis encompass a combination of clinical presentations, risk assessments, and patient-specific factors: •Clinical Presentations: While asymptomatic cases remain a challenge, commonly encountered symptoms such as vaginal or urethral discharge, itching, pain during intercourse, or post-coital bleeding immediately warrant testing.
•Risk Assessment: Patients with a history of multiple sexual partners, inconsistent condom use, or a known contact with an infected individual are considered high-risk and are thus recommended for testing.
•Patient-specific Factors: Pregnant women are routinely tested due to the potential complications T. vaginalis can pose during pregnancy.Additionally, individuals with a history of recurrent STIs or those presenting with other STI symptoms are also prioritized for testing to rule out co-infections or a misdiagnosed recurrent infection.
•Other Triggers: Given its 'clinical chameleon' nature, any unexplained or persistent reproductive tract symptoms, especially if other tests come back negative, serve as a trigger for T. vaginalis testing as a diagnostic precaution.
By adhering to these protocols, we aim to capture a broad spectrum of potential T. vaginalis infections, ensuring that even subtle presentations don't go undetected.
This has been added into the revised manuscript from line numbers 146 to 165.
8. Despite these non-specific presentations, there is no description of other diagnostic tests or investigations conducted e.g.chlamydia or gonorrhoea NAAT, microscopy for clue cells, syphilis or HIV testing.These should be included with results to ensure no evidence of co-infection.

Co-testing Protocols and Outcomes:
Recognizing the potential for co-infections and the overlapping symptomatology of various STIs with Trichomonas vaginalis, we employed a multi-test diagnostic approach in all presented cases: •Microscopy for Clue Cells:To rule out bacterial vaginosis, microscopic examinations were conducted to detect the presence of clue cells.None of the female patients exhibited clue cells on their wet mount microscopy.
•Syphilis Testing:Given the risk and implications of untreated syphilis, all patients underwent a Rapid Plasma Reagin (RPR) test.
All tests returned non-reactive, indicating no active syphilis infections among the patients.
•HIV Testing:Considering the enhanced risk of HIV transmission associated with STIs, HIV tests were also conducted for each patient.All tested negative.
While our facility does not offer Chlamydia and Gonorrhoea NAATs, we emphasize clinical evaluation and other available tests to achieve a comprehensive diagnostic picture.This thorough approach ensures an understanding of the broader STI status of the patient, paving the way for informed treatment decisions.
This has been added to the revised manuscript and reflects in line numbers 207 to 222. 9.There is inconsistent reporting of treatment protocols -ranging from a single dose metronidazole to 1 week of therapy.Please provide clearer overview of treatment administered, the rationale, and which treatment guidelines are being followed.

Treatment Protocols for Trichomonas vaginalis:
The choice of treatment regimen for T. vaginalis at our facility is dictated by several factors, including the patient's clinical presentation, co-existing medical conditions, and specific circumstances.Below is an overview of our treatment protocols and the rationale for each: •Standard Treatment: The primary treatment for uncomplicated T. vaginalis infection in non-pregnant individuals is a single dose of oral metronidazole.This approach is backed by its high efficacy, convenience for the patient, and compliance assurance.
•Extended Treatment: In cases where symptoms are more severe, persistent, or where there's a history of recent treatment failures, a 7-day regimen of metronidazole is preferred.This extended duration ensures complete eradication of the infection and minimizes the chances of resistance or recurrence.
•Pregnant Patients: Given the potential risks associated with oral metronidazole during pregnancy, especially in the first trimester, topical metronidazole gel is our go-to option.This local treatment effectively manages the infection while minimizing systemic exposure to the drug.
•Treatment Guidelines: Our protocols align with the treatment recommendations set forth by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC).Both these guidelines emphasize tailoring the treatment approach based on the individual's clinical and demographic factors.
By adhering to these standardized protocols and guidelines, we ensure that each patient receives optimal care tailored to their specific needs, ensuring the highest chances of treatment success.
The same has been added to the revised manuscript from line numbers 223 to 245.
10. Diagnostic stewardship refers to utilising the correct diagnostic test, for the correct patient, in the correct circumstances.Please expand on how you feel microscopy achieves this goal, given the manuscript currently suggests that this will be undertaken for all patients irrespective of symptoms.

Microscopy within Diagnostic Stewardship:
The ethos of diagnostic stewardship is to ensure the right test is given to the right patient at the right time.Microscopy, especially wet mount microscopy for the detection of Trichomonas vaginalis, aligns with these principles in the following ways: •Rapid and Reliable: Microscopy provides an immediate visualization of T. vaginalis trophozoites, offering swift diagnosis.Its accuracy, when performed correctly, ensures that patients receive timely and appropriate treatment.
•Cost-effective: Given the resource constraints in many healthcare settings, microscopy serves as an affordable diagnostic tool.It does not require advanced equipment or reagents, making it accessible and sustainable.
•Targeted Testing: While our manuscript may give the impression that microscopy is conducted universally, in practice, it is administered based on clinical judgment.Patients presenting with symptoms consistent with trichomoniasis or those identified as high-risk through our screening protocols are prioritized for microscopic evaluation.
•Complement to Other Tests: In cases where microscopy results are inconclusive or if co-infections are suspected, microscopy can act as a stepping stone to more specific tests.It aids in narrowing down differential diagnoses, ensuring that subsequent tests are more focused and judiciously used.
•Reduction in Unnecessary Treatment: Accurate diagnosis through microscopy reduces the chances of treating non-existent infections.This not only prevents unnecessary drug exposure for patients but also aids in antimicrobial stewardship, reducing the chances of resistance development.
By integrating microscopy within our diagnostic approach, and using it judiciously based on clinical indicators, we align with the principles of diagnostic stewardship.It ensures that our patients receive optimal care, diagnostics are cost-effective, and resources are utilized efficiently.
This has been added in the revised manuscript and reflects in the line numbers 317 to 343.

Training Intervention and Its Transformative Impact:
To address recurring challenges in sample quality and unnecessary test requisitions, we orchestrated a targeted interactive session with our Obstetrics and Gynecology colleagues in the month of May 2023.This initiative was driven by the increasing influx of High Vaginal Swabs (HVS) without discernible clinical relevance.

Content of the Training:
Our training was structured around key themes to ensure a comprehensive understanding: •Proper Sample Collection: Emphasized the critical role of obtaining high-quality samples, discussing the ideal techniques, and common pitfalls to avoid.
•Wet Mount Microscopy: Introduced its significance in the rapid and accurate diagnosis of infections like Trichomoniasis, illustrating its practical application.
•Gram Staining: Demonstrated its pivotal role in discerning various infections, particularly in distinguishing between bacterial vaginosis, candidiasis, and trichomoniasis.
•Questioning the Necessity of Culture: Given that many causative agents of bacterial vaginosis are anaerobes and both candidiasis and trichomoniasis can be effectively diagnosed with Gram staining and wet mount, we questioned the routine need for culture.This segment emphasized the judicious use of culture, conserving resources, and ensuring timely diagnoses.

Post-training Impact:
The repercussions of this training were multifaceted and immensely positive with most of our cases reporting positive post training: •Decrease in Unnecessary Requests: There was a marked reduction in superfluous culture and sensitivity requests.This not only conserved resources but also ensured that laboratory efforts were channeled into clinically relevant tests.
•Enhanced Understanding Among Gynecologists: The session armed the OBG specialists with insights into proper sample collection and relevant test requisition.Their newfound knowledge ensured that samples sent were of optimal quality and that the requested tests aligned with the patient's clinical presentation.
•Collaborative Synergy: This initiative fostered a collaborative environment, bridging the gap between laboratory specialists and clinicians.This synergy promises better patient outcomes, with both teams being well-informed and aligned in their approach.
In essence, through this targeted training session, we successfully optimized our diagnostic approach, ensuring efficient utilization of resources and fostering a collaborative spirit between laboratory specialists and clinicians.
The same has been added to the revised manuscript from line numbers 259 to 293.
12. Finally, the suggested peer reviewers appear to also be affiliated with the All India Institute of Medical and would be inappropriate to act as reviewers.Please suggest reviewers from alternative institutions, who you have not previously published or worked with.
Names of new reviewers not belonging to All India Institute of Medical Sciences has been suggested in the resubmission.Major revisions will need to be made before we can consider sending your manuscript for external peer review.Specific comments from the editorial board which should be made prior to resubmission are highlighted in comments below: -This manuscript presents a case series of 5 cases of Trichomonas vaginalis infections and reports a change to local diagnostic and sampling protocols -Whilst it is implied within the manuscript that the key change has been to shift from standard culture to wet mount microscopy, this is not explicitly or clearly described.Please include a paragraph which clearly articulates what the previous standard diagnostic protocols were, and how they have now been changed.-Similarly, the manuscript makes repeated reference to improving 'proper sampling', however there is no description as to what the authors mean in relation to sampling standards.How have these changed and what is the new standard?-The abstract states the authors have conducted a comprehensive literature review, however this is not apparent in the main body of the manuscript.A comprehensive literature review of TV infections should be performed or at least referenced int he linked literature.-The authors state disease burden is high however offer no epidemiological data to corroborate estimates of TV incidence or prevalence.-The introduction states 5 cases of female TV infections will be presented, then 4 females and 1 male are discussed in the main body of the manuscript.-It is unclear as to the timescale over which these patients were identified.Please clearly include dates of the cases in order to strengthen your assertion that the diagnoses were a result of changes in diagnostic practices.-The authors report that TV is a 'clinical chameleon', insinuating that this diagnosis cannot be reached on the basis of clinical history and examination findings alone.There is a spectrum of clinical presentations reported ranging from asymptomatic carriage to vaginal/urethral discharge and bleeding -please comment on what protocols are in place to trigger testing for TV -is there a specific set of symptoms, risk profile, patient characteristics?-Despite these non-specific presentations, there is no description of other diagnostic tests or investigations conducted e.g.chlamydia or gonorrhoea NAAT, microscopy for clue cells, syphilis or HIV testing.These should be included with results to ensure no evidence of co-infection.-There is inconsistent reporting of treatment protocols -ranging from a single dose metronidazole to 1 week of therapy.Please provide clearer overview of treatment administered, the rationale, and which treatment guidelines are being followed.-Diagnostic stewardship refers to utilising the correct diagnostic test, for the correct patient, in the correct circumstances.Please expand on how you feel microscopy achieves this goal, given the manuscript currently suggests that this will be undertaken for all patients irrespective of symptoms.-There is limited discussion of what the training package entailed.Please elaborate on who this training was directed to, what did it include, what were the overarching goals, and how has this has impacted practice.Are there quantifiable measures to assess practice before and after implementation of the training?-Finally, the suggested peer reviewers appear to also be affiliated with the All India Institute of Medical Sciences and would be inappropriate to act as reviewers.Please suggest reviewers from alternative institutions, who you have not previously published or worked with.If you are able to address the above concerns, we would be happy to arrange external peer review upon re-submission.Thank you for submitting to Access Microbiology.

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Patel et al 2006) does not support 7% figure nor any specific reference to Gujarat -please review Response: In a separate study conducted in Surat, Gujarat, out of 102 samples, 35 were positive for Trichomonas vaginalis by the culture method.(Chakraborty T, Mulla SA, Kosambiya JK, Desai VK.Prevalence of Trichomonas vaginalis infection in and around Surat.Indian J Pathol Microbiol.2005 Oct;48(4):542-5.PMID: 16366122).

vERSIOn 1 Editor
recommendation and comments https://doi.org/10.1099/acmi.0.000698.v1.3 © 2023 Smith C.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Christopher Smith; Imperial College London, UNITED KINGDOM Date report received: 12 September 2023 Recommendation: Major Revision Comments: Thank you for your submission to Access Microbiology.The editorial board has now considered your article 'Case Series on Trichomonas vaginalis Infections: Impact of Proper Sample Collection and Diagnostic Stewardship' and awarded the following outcome: Major Revision Required.

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Correct sampling site.Depending on the patient's symptoms and gender, emphasizing the correct anatomical site for sample collection.•Avoiding contamination.Ensuring that samples are free from contaminants, including lubricants or douches, which can interfere with microscopy.•

Volume and technique. Adequate
sample volume and correct swabbing technique to ensure a representative sample.
(11)ensuring adequate sample volume with swabs, our team focused on training healthcare professionals in proper swabbing techniques.This included the correct insertion depth, duration of contact with the mucosal surface, and rotation of the swab to ensure sufficient sample collection.This has been added to the revised manuscript and is reflected in line numbers 215 to 218.A study conducted in Mysore, India, found an 8.5% prevalence of Trichomonas vaginalisamong young reproductive-aged women (Madhivanan P, Bartman MT, Pasutti L, Krupp K, Arun A, Reingold AL, Klausner JD.Prevalence of Trichomonas vaginalis infection among young reproductive age women in India: implications for treatment and prevention.PubMed.2009Nov;8(11):902-7. doi: 10.1071/SH09015).
202, 203•Volume and Technique: Adequate sample volume and correct swabbing technique toensure a representative sample.Response: article "Diagnosis and Management of Trichomonas vaginalis" (Kissinger PJ, Gaydos CA, Seña AC, et al.Diagnosis and Management of Trichomonas vaginalis: Summary of Evidence Reviewed for the 2021 Centres for Disease Control and Prevention Sexually Transmitted Infections Treatment Guidelines.Clin Infect Dis.2022 Apr 13;74(Suppl_2):S152-S161.doi: 10.1093/cid/ ciac030)provides a summary of evidence reviewed for the 2021 Centres for Disease Control and Prevention (CDC) Sexually Transmitted Infections Treatment Guidelines.It mentions the utility of newly available diagnostic methods, such as point-of-care assays and multiple nucleic acid amplification tests (NAATs) .
This has been added to the reference section of the manuscript and is reflected as reference number 10.This is a study that would be of interest to the field and community.However, there are important edits required as highlighted by peer-reviewers -specifically related to referencing.Please address these significant concerns before further consideration can be given to publication.Overall it's well written and the subject matter is important.There are issues with the references which should be reviewed and an incorrect assertion that metronidazole is unsafe in pregnancy which must be corrected.-heading Intro-Jos University Teaching Hospital, Medical Microbiology, Medical Microbiology Dept.Jos University Teaching Hosp, #1 Alex Kazen Close Lamingo Jos North Plateau State, Jos, NIGERIA https://orcid.org/0000-0003-2694-7596Date report received: 12 November 2023 Recommendation: Minor Amendment Comments: 1. Comment I would expect that as part of timely processing of samples, sometimes a mini side lab equiped with basic microscopy equipment and personnel is deployed to achieve this and also reduce GU samples competing with general lab samples.So how did you achieve timely transportation and processing?196 Timely Processing: Ensuring that samples are transported to the laboratory and 197 processed as swiftly as possible to maintain organism viability.2. Comment How can sample volume be termed adequate in samples taken with swabs?202 Volume and Technique: Adequate sample volume and correct swabbing technique to 203 ensure a representative sample.
9 (Luther et al. 2018) does not support the statement made about positive impact on training interventions.Please review.Response: The reference 9 (Luther VP, Shnekendorf R, Abbo LM, et al.Antimicrobial Stewardship Training for Infectious Diseases Fellows: Program Directors Identify a Curriculum Need.Clin Infect Dis.2018;67(8):1285-1287) has been removed.The study "Improving sexually transmitted infection screening, testing, and treatment among people with HIV" highlights the necessity of provider training."(Cullinen K, Hill M, Anderson T, Jones V, Nelson J, Halawani M, et al. (2021) Improving sexually transmitted infection screening, testing, and treatment among people with HIV: A mixed method needs assessment to inform a multi-site, multi-level intervention and evaluation plan.PLoS ONE 16(12): e0261824.https://doi.org/10.1371/journal.pone.0261824)shows that gaps in conducting consistent sexual histories can influence the frequency of STI testing and underscores the importance of comprehensive sexual health histories in routine care, which is often lacking.The study supports the implementation of evidence-based interventions such as provider training related to bacterial STI screening, testing, treatment, and follow-up, to address these gaps .duction: 1. Reference 2 (Das et al 2011) does not support 2-10% figure -please review 2. Reference 3 (Patel et al 2006) does not support 7% figure nor any specific reference to Gujarat -please review -heading a) Traditional Diagnostic Approaches: 3. Reference 1 (Schwebke et al 2004) does not support suggestion sensitivity of microscopy is as low as 50% -they state 60-70% sensitivity.Please review.-headingCase presentation: 4. Please specify metronidazole doses used and strength of topical treatments. 5.In case 3 -please remove the following sentence as it incorrectly implies oral metronidazole is unsafe in pregnancy: "Considering her pregnancy, the patient was treated with topical metronidazole gel to avoid potential risks associated with oral medication" 6. Cases 4 and 5 have no treatment mentioned.please review.heading Shift in Diagnostic Protocols 7. Reference 7 (Ali and Nozaki 2007) is a review article about therapeutics for parasites.It contains nothing about diagnostic processes.Please review.-heading Treatment Protocols for Trichomonas vaginalis: 8. Please state doses used for metronidazole.9. Pregnant patients: There is no evidence of teratogenicity from use of oral metronidazole during any stage of pregnancy.However, there is evidence that TV causes serious complications in pregnancy such as pre-term delivery and low birth weight.The efficacy of topical therapeutics for TV are uncertain at best -conflicting results in the literature, some suggesting topical metronidazole is inferior to systemic therapy.This is reflected in the WHO guideline you mention whereby they advise topical metronidazole is not recommended for TV and that oral metronidazole is recommended for treatment of TV in pregnancy (Sherrard, IUSTI/WHO management of vaginal discharge 2018).As such, I cannot condone suggesting to the reader that topical treatment of TV in pregnancy is sufficient.10.Please provide references for WHO and CDC guidelines.-heading 2. The Role of Sensitization and Training: 11.Reference 8 (Patel AL et al.Clin Infect Dis 2021; 73(Suppl 1):S55-61) -this paper does not exist.Please review.12. Reference 9 (Luther et al. 2018) does not support the statement made about positive impact on training interventions.Please review.https://doi.org/10.1099/acmi.0.000698.v2.4 © 2023 Alobu E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Emeka Walter Alobu; 11.There is limited discussion of what the training package entailed.Please elaborate on who this training was directed to, what did it include, what were the overarching goals, and how has this has impacted practice.Are there quantifiable measures to assess practice before and after implementation of the training?